Mycoplasma is a term used to denote a species included in the class Mollicutes. They are the smallest and simplest free-living parasitic organisms known. Mycoplasma are parasites of many animal species and typically exhibit host and tissue specificity. In humans, Mycoplasma (M.) pneumoniae is the respiratory pathogen responsible for atypical pneumonia. Mycoplasma are frequently isolated from patients with immunodeficiencies associated with disease states.
Mycoplasma are common contaminants of eukaryotic cell cultures and are known to alter the phenotypic characteristics of host cell lines. The published incidence of mycoplasma infected cell cultures has ranged from 4 to 92%. Of the 18 most common species recognized as culture contaminants, M. orale, M. hyorhinis, M. arginini, M. fermentans, and Acholeplasma (A.) laidlawii are the most frequently isolated representing 80 to 90% of all isolates. The small size of mycoplasma allows them to pass through the commonly used 0.45 μm sterilization filters and mycoplasma are typically resistant to antibiotics such as penicillin and streptomycin. Mycoplasma contamination usually does not produce visible changes in cell culture medium despite the fact it can reach titers of 108 per milliliter. Sources of mycoplasma contamination include laboratory personnel, reagents, and mycoplasma contaminated cell lines.
Mycoplasma contamination is detected by a number of methods. Microbial culture is generally considered the most sensitive method for mycoplasma screening and is commonly used as a reference for the evaluation of any new mycoplasma detection techniques. However, culturing mycoplasma can take two to four weeks, and cannot detect fastidious mycoplasma. Moreover, culturing mycoplasma requires special growth conditions and is generally restricted to specialized laboratories.
Another method involves microscopic visualization of mycoplasma attached to host cells using fluorescent DNA staining that displays extra-cellular fluorescence as well as extra-nuclear fluorescence. This method is efficient for mycoplasma screening and particularly for detection of M. hyorhinis strains that cannot be cultivated on microbiological media. However, fluorescent staining cannot detect plasma species that cyto-absorb poorly. Moreover, this method requires expertise for accurate interpretation of results.
Enzyme-linked immunosorbent assays measuring mycoplasma specific cell-surface antigens have been described but typically lack sensitivity. A number of polymerase chain reaction (PCR) based methods have also been described for the detection of mycoplasma that achieve high sensitivity and may be amenable to species identification. However, PCR-based detection of mycoplasma is prone to false positives due to amplicon contamination and false negative results due to use of excess sample. Mycoplasma screening methods utilizing select biochemical activity have also been used, but give inconsistent results when comparing different cell lines.
While these known methods have provided researchers with various techniques to determine mycoplasma contamination, these techniques have several disadvantages as described above. More efficient, rapid and sensitive methods of detecting contamination are desired.